Microscope apparatus

ABSTRACT

Providing a microscope apparatus capable of observing phase variation of a phase object with sufficient contrast with a white light source having an area such as a halogen lamp and a mercury lamp commonly used in a microscope. A microscope apparatus  100  comprising: an illumination optical system  10  that illuminates a sample  5  with illumination light emitted from a light source  1;  an imaging optical system  30  that converges light emitted from the sample to form a sample image by an objective lens  6;  an aperture member  3  that is disposed in the illumination optical system in the vicinity of a conjugate plane of a rear focal plane of the objective lens and has an aperture for limiting the illumination light; and a filter member that includes a phase plate  7  that is disposed in the imaging optical system in the vicinity of the rear focal plane of the objective lens or in the vicinity of the conjugate plane of the rear focal plane of the objective lens and has a first phase area introducing phase difference of 180 degrees into the light from the sample and a second phase area; a phase boundary portion between the first phase area and the second phase area being disposed in a conjugate aperture of the aperture.

TECHNICAL FIELD

The present invention relates to a microscope apparatus.

BACKGROUND ART

An object to be observed by a microscope is mainly divided into twocategories, in which one is an amplitude object and the other is a phaseobject. Since the amplitude object varies brightness or colors thereof,the variation can be detected as a contrast by an eye or an imagingdevice such as a CCD. On the other hand, since a phase object onlyvaries phase of light, it shows a poor contrast and hardlydistinguishable as it is. Accordingly, there has been proposed variousmethods for changing phase variation of a phase object into discerniblevariation in contrast such as Japanese Patent Application Laid-Open No.11-95174.

However, in examples disclosed in Japanese Patent Application Laid-OpenNo. 11-95174, in order to make phase variation of a phase objectdiscernible variation in contrast, a light source is limited to acoherent light source.

DISCLOSURE OF THE INVENTION

The present invention is made in view of the above-described problemsand has an object to provide a microscope apparatus capable of observingphase variation of a phase object with sufficient contrast with a whitelight source having an area such as a halogen lamp and a mercury lampcommonly used in a microscope.

According to a first aspect of the present invention, there is provideda microscope apparatus comprising: an illumination optical system thatilluminates a sample with illumination light emitted from a lightsource; an imaging optical system that converges light emitted from thesample to form a sample image by an objective lens; an aperture memberthat is disposed in the illumination optical system in the vicinity of aconjugate plane of a rear focal plane of the objective lens and has anaperture for limiting the illumination light; and a filter member thatincludes a phase plate that is disposed in the imaging optical system inthe vicinity of the rear focal plane of the objective lens or in thevicinity of the conjugate plane of the rear focal plane of the objectivelens and has a first phase area introducing phase difference of 180degrees into the light from the sample and a second phase area; a phaseboundary portion between the first phase area and the second phase areabeing disposed in a conjugate aperture of the aperture.

In the first aspect of the present invention, it is preferable that theaperture is a slit aperture, and the phase boundary portion issubstantially parallel to a long sides direction of the slit aperture.

In the first aspect of the present invention, the following conditionalexpression (1) is preferably satisfied:

0.05≦d1/(2×NA×f×m)≦0.6  (1)

where d1 denotes a short side width of the slit aperture, NA denotes anumerical aperture of the objective lens, f denotes a focal length ofthe objective lens, and m denotes magnification from the rear focalplane of the objective lens to the plane in the illumination opticalsystem where the slit aperture is disposed.

In the first aspect of the present invention, it is preferable that thefilter member further includes a transmittance controlling plate thatcontrols transmittance of the phase plate at the conjugate position ofthe slit aperture, and the transmittance controlling plate hassubstantially constant transmittance over the conjugate position of theslit aperture, and transmittance t preferably satisfies the followingconditional expression (2):

0≦t≦50 (unit: %)  (2).

In the first aspect of the present invention, it is preferable that, inthe phase plate, when the phase boundary is assumed to be the Y axis,the axis perpendicular to the Y axis and the optical axis is X axis, andthe point of intersection of the X axis and the Y axis is to be anorigin, the filter member further includes a transmittance controllingplate that has transmittance distribution such that transmittance of thefilter member becomes minimum at the origin, and higher as being awayfrom the origin, and the transmittance distribution is symmetrical withrespect to the Y axis.

In the first aspect of the present invention, it is preferable that, inthe phase plate, when the phase boundary is assumed to be the Y axis,the axis perpendicular to the Y axis and the optical axis is X axis, andthe point of intersection of the X axis and the Y axis is to be anorigin, the filter member further includes a transmittance controllingplate that has transmittance distribution such that transmittance of thefilter member becomes higher step-by-step as being away from the origin,and the transmittance distribution is symmetrical with respect to the Yaxis.

In the first aspect of the present invention, it is preferable that theaperture of the aperture member is an annular aperture, the phaseboundary portion of the phase plate is circular, and the phase boundaryportion is disposed substantially the center of an annular apertureconjugate with the annular aperture.

In the first aspect of the present invention, the following conditionalexpression (3) is preferably satisfied:

0.025≦d2/(2×NA×f×m)≦0.6  (3)

where d2 denotes an aperture width of the annular aperture, NA denotes anumerical aperture of the objective lens, f denotes a focal length ofthe objective lens, and m denotes magnification from the rear focalplane of the objective lens to the plane in the illumination opticalsystem where the annular aperture is disposed.

In the first aspect of the present invention, it is preferable that thefilter member further includes a transmittance controlling plate thatcontrols transmittance of the phase plate at the conjugate position ofthe annular aperture, and the transmittance controlling plate hassubstantially constant transmittance over the conjugate position of theannular aperture, and transmittance t preferably satisfies the followingconditional expression (2):

0≦t≦50 (unit: %).

In the first aspect of the present invention, it is preferable that thefilter member further includes a transmittance controlling plate thathas transmittance distribution concentric with respect to the opticalaxis, and the concentric transmittance distribution is such thattransmittance becomes minimum at an annular aperture positionsubstantially conjugate with the annular aperture of the phase plate,becomes higher step-by-step as being away from the annular apertureposition substantially conjugate with the annular aperture of the phaseplate, and is substantially symmetrical between a direction from aninner circumference of the aperture conjugate with the annular apertureto the center direction of the annular aperture and a direction from anouter circumference of the aperture conjugate with the annular apertureto the outer direction.

In the first aspect of the present invention, it is preferable that thefilter member includes a plurality of phase plates and a plurality oftransmittance controlling plates, and the plurality of phase plates andthe plurality of transmittance controlling plates are independentlychangeable with respect to the optical axis.

In the first aspect of the present invention, it is preferable that thefilter member is changeable with respect to the optical axis.

The present invention makes it possible to provide a microscopeapparatus capable of observing phase variation of a phase object withsufficient contrast with a white light source having an area such as ahalogen lamp and a mercury lamp commonly used in a microscope.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is schematic diagram showing a microscope apparatus according toa first embodiment of the present invention.

FIGS. 2A, 2B and 2C are diagrams showing a π phase plate disposed in themicroscope apparatus according to the first embodiment, in which FIG. 2Ashows construction and positional relation a slit aperture, FIG. 2Bshows transmittance characteristics of the π phase plate, and FIG. 2Cshows phase characteristics of the π phase plate.

FIG. 3 is a graph showing a response function of the π phase plate witha phase characteristic shown in FIG. 2C.

FIGS. 4A through 4D are graphs showing imaging simulation results uponvarying the value of x where σ=0 in the π phase plate shown in FIG. 2A.

FIGS. 5A through 5D are graphs showing imaging simulation results uponvarying the value of σ where x=0 in the π phase plate shown in FIG. 2A.

FIG. 6 is a graph showing contrast upon varying the value of σ where x=0in the π phase plate shown in FIG. 2A in comparison with bright fieldobservation.

FIGS. 7A, 7B and 7C are diagrams showing a π phase plate with a filterhaving transmittance t disposed in a microscope apparatus according to asecond embodiment, in which FIG. 7A shows construction and positionalrelation of a slit aperture, FIG. 7B shows transmittance characteristicsof the π phase plate, and FIG. 7C shows phase characteristics of the πphase plate.

FIGS. 8A through 8F are graphs showing imaging simulation results uponvarying the values of x and t where σ=0.1 in the π phase plate shown inFIG. 7A.

FIGS. 9A, 9B and 9C are diagrams showing a π phase plate disposed in amicroscope apparatus according to a third embodiment, in which FIG. 9Ashows construction and positional relation of an annular slit aperture,FIG. 9B shows transmittance characteristics of the π phase plate, andFIG. 9C shows phase characteristics of the π phase plate.

FIGS. 10A, 10B and 10C are diagrams showing a variation of the thirdembodiment, in which FIG. 10A shows construction and positional relationof an annular aperture with a filter having transmittance t, FIG. 10Bshows transmittance characteristics of the π phase plate, and FIG. 10Cshows phase characteristics of the π phase plate.

FIG. 11 is a schematic diagram showing a microscope apparatus accordingto a fourth embodiment of the present invention.

FIGS. 12A and 12B are diagrams respectively showing an example of aslider type aperture member and a slider type phase plate holder used inthe fourth embodiment.

FIG. 13 is a diagram showing a variation of the microscope according tothe fourth embodiment.

FIGS. 14A, 14B and 14C are diagrams showing the variation of the fourthembodiment, in which FIG. 14A is a slider type filter member, FIG. 14Bis a slider type π phase plate, and FIG. 14C is a combination of FIGS.14A and 14B.

THE BEST MODE FOR CARRYING OUT THE INVENTION

Each embodiment according to the present invention is explained withreference to accompanying drawings. In the following embodiment, theembodiment is explained with a transmission type microscope.

First Embodiment

FIG. 1 is schematic diagram showing a microscope apparatus according toa first embodiment of the present invention.

In FIG. 1, illumination light emitted from a white light source 1 suchas a halogen lamp or a mercury lamp is converged by a collector lens 2,transmitted through a slit member 3 having a slit aperture 3 a, andilluminates a sample 5 by an illumination optical system 10 including acondenser lens 4. Light transmitted through the sample 5 is converged byan objective lens 6, transmitted through a π phase plate 7 giving aphase difference of 180 degrees, and forms a sample image on an imageplane 8 through an imaging optical system 30.

The π phase plate 7 is disposed in the vicinity of a rear focal plane ofthe objective lens 6, and the slit member 3 is disposed in the vicinityof a front focal plane of the condenser lens 4, which is a conjugateplane with the π phase plate 7. Here, the rear focal plane of theobjective lens 6 and the front focal plane of the condenser lens 4 areconjugate planes with each other. The π phase plate 7 may be disposed inthe vicinity of a conjugate plane with the rear focal plane of theobjective lens 6 in the imaging optical system 30. The slit member 3 maybe disposed in the vicinity of a conjugate plane with the front focalplane of the condenser lens 4 in the illumination optical system 10.

Here, a Z axis is assumed in the direction of the optical axis, and XYaxis is assumed on a plane perpendicular to the optical axis. The πphase plate 7 is movable in the XYZ directions. The movement in Zdirection is set for corresponding variation in the position of the rearfocal plane of the objective lens 6 upon changing the objective lens 6.Movement in XY directions are used for adjusting alignment of the πphase plate 7, and for adjusting a contrast upon eye observation or uponobtaining images by an imaging device (not shown). In this manner, themicroscope apparatus 100 is constructed.

FIGS. 2A, 2B and 2C are diagrams showing a π phase plate disposed in themicroscope apparatus according to the first embodiment. FIG. 2A showsconstruction and positional relation of a slit aperture seen from anarrow A in FIG. 1 and XY axes, which are perpendicular to the opticalaxis, are perpendicular to each other and included in the plane of the πphase plate 7. In FIG. 2A, an outer circle 7 a denotes an effectivediameter of an objective pupil of the objective lens 6, and theeffective diameter of the objective pupil is normalized by x=1 and y=1.Such normalization is applied to other embodiments. A rectangular solidline in FIG. 2A shows a conjugate aperture of the slit aperture 3 a ofthe slit member 3, which is a projected image of the slit aperture 3 aon the plane of the π phase plate 7 and is denoted by the same symbol of3 a. FIG. 2B shows transmittance distribution of the π phase plate inthe X axis direction corresponding to the objective pupil diameter. FIG.2C shows phase distribution of the π phase plate 7. The π phase plate 7has a phase plate 7 e having a phase difference of −π/2 on the −X sidewith respect to the Y axis, and a phase plate 7 f having a phasedifference of +π/2 on the X side with respect to the Y axis, and a casethat the phase boundary 7 c, which is the boundary of the both plates,is coincide with the Y axis is shown. In this case, the phase plate 7 ehas a phase difference of −π/2 and the phase plate 7 f has a phasedifference of +π/2, and the case the total phase difference is π isexplained. However, the present embodiment is not limited to theabove-described condition, and it is sufficient that light from thesample passing through the phase plate 7 f has a phase difference of π(180 degrees) with respect to light from the sample passing through thephase plate 7 e. Moreover, the reason why the π phase plate 7 hastransmittance distribution or phase distribution having a value otherthan 0 on the outside of the outer circle 7 a is that the effectivediameter of the objective pupil of the objective lens 6 is not blockedupon moving the π phase plate 7 in XY directions.

Then, the imaging simulation is explained.

Phase distribution F(x) in X axis direction within the outer circle 7 ashown in FIG. 2C is shown by the following expression (a1):

$\begin{matrix}{{{F(x)} = {i \cdot {{sgn}(x)}}}{where}\begin{matrix}{{{{sgn}(x)} = 1},{0 < x \leq 1}} \\{{= 0},{x = 0}} \\{{= {- 1}},{{- 1} \leq x < 0.}}\end{matrix}} & \left( {a\; 1} \right)\end{matrix}$

Expression (a1) is a transfer function in a frequency space ofone-dimensional Hilbert transform. In phase distribution shown in FIG.2C, since phase difference is π at the boundary x=0, approximately ahalf of light transmitting through the sample 5 having phasedistribution receives phase shift π, and reaches the image plane 8 tomake interference. As a result, phase distribution of the sample 5becomes visible as intensity distribution on the image plane 8.

How does phase distribution on the sample 5 become visible as a sampleimage on the image plane 8 is explained below.

As a matter of simplicity, one-dimension (X axis direction) is to beconsidered. Moreover, the slit aperture 3 a is assumed to be aninfinitesimal pinhole. Amplitude distribution of the sample 5 upon beingilluminated with a point source is s(x′), and Fourier transform thereofis S(x). Amplitude distribution of the sample image on the image plane 8is g(x′), and Fourier transform thereof is G(x). Then, S(x), G(x), andF(x) are shown by the following expression (a2):

G(x)=S(x)·F(x)  (a2)

In this case, when the sample 5 is assumed to be a weak phase object,

s(x′)=exp(iφ(x′))≈1+iφ(x′)  (a3)

and diffracted light from the sample 5 is given by Fourier transformthereof.

S(x)=δ(x)+Φ(x)  (a4)

where Φ(x) denotes Fourier transform of φ.

Substituting this into expression (a2), phase distribution componentΦ(x) is left.

G(x)=Φ(x)·F(x)  (a5)

Since F(x) is a transfer function in a frequency space of Hilberttransform, assuming that φH(x′) is Hilbert transform of φ(x′), amplitudedistribution of the image g(x′) is

g(x′)=φH(x′)  (a6).

Intensity distribution is

|g(x′)|² =φH(x′)²  (a7).

When this is moved to the image space, convolution of s(x′), whichcorresponds distribution that the sample 5 is illuminated by a pointsource, and inverse Fourier transform of f(x′) as a point spreadfunction becomes g(x′) shown in the following expression (a8):

g(x′)=s(x′)*f(x′)  (a8)

where “*” denotes convolution.

FIG. 3 is a graph showing f(x′). As shown in FIG. 3, point spreadfunction in Hilbert transform has a contrast to a phase object, and theshape of the contrast has a contrast shape, and the contrast shape showsa so-called differential image.

Imaging simulation results by an ideal lens are shown below. Simulationcondition is in a biological observation using a general purposeobjective lens with a magnification of 40, and coherency of theillumination light a is expressed by the following expression (0):

σ=d1/(2×NA×f×m)  (0)

where d1 denotes an aperture width that limits illumination light, andcorresponds to the slit width d1 in FIG. 2A.

In expression (0), a numerical aperture of the objective lens 6 isNA=0.6, the focal length of the objective lens 6 is f=5 mm, and themagnification from the rear focal plane of the objective lens 6 to theplane where the slit member 3 is disposed is m=1.

The sample is assumed to have transmittance=1 (100%), phase difference100 nm, width W=100 μm (converted on the image plane), and a rectangularshape, and to be disposed at the center of the visual field (x=0).Observation wavelength λ=588 nm.

FIGS. 4A through 4D shows a simulation result where σ=0 in expression(0), in other words, the light source 1 is assumed to be a coherentlight source. Here, σ=0 means the aperture width d1 is assumed to beinfinitesimal, and does not mean d1=0. Moreover, when the coherent lightsource σ=0 is used, the slit member 3 is not necessary, so that thepresent invention becomes meaningless to this case. FIGS. 4A through 4Dshow the imaging simulation results. FIG. 4A corresponds to the casethat the phase boundary 7 c of the π phase plate 7 comes to the opticalaxis (origin: x=0), and FIGS. 4B through 4D are imaging simulationresults corresponding to the cases the position of the π phase plate 7is shifted to the X direction by 0.2 mm each. FIG. 4A shows a contrastimage similar to a so-called differential image, however it is noisyimage. For example, x value is −50 μm or less, and +50 μm or more, thebackground signal becomes noisy with a wave shape. As shown in FIGS. 4Bthrough 4D, when the π phase plate 7 is shifted to the X direction, acontrast image similar to a so-called pseudo-relief image is obtained,however undulation with a frequency corresponding to the shift amount issuperposed on the background signal. This is because the shift in the Xdirection of the π phase plate 7 makes frequency modulationcorresponding to the shift amount. It is undesirable for a microscopeimage that such an undulation component is superposed on the backgroundsignal.

In this manner, when σ=0 in other words the light source 1 is a coherentlight source, it is difficult to obtain excellent contrast image.

Then, in the present invention, imaging simulation results upon varyingσ such that σ=0.05, 0.1, 0.2 (corresponding to the cases varying theslit width d1) at x=0 are shown in FIGS. 5A through 5C. As shown inFIGS. 5A through 5C, by disposing the slit member 3 having a slitaperture 3 a with each slit width d1 in the illumination optical system10, although contrast becomes lower than the case the coherent lightsource (σ=0, see FIG. 4A), noise on the background signal becomes low,and characteristic of a so-called differential image becomes good.

FIG. 5D is a graph showing an imaging simulation result where σ=0.1,x=0.3 mm. By shifting in the X direction, although the contrast forms aso-called pseudo-relief image, the undulation of the frequencymodulation component does not superpose on the background signal incomparison with FIG. 4C, and almost flat background signal can beobtained.

This is because the frequency modulation components are accumulatedwithin the range of the slit width d1 and averaged. According to thecalculation, this effect is insufficient upon σ=0.05 shown in FIG. 5A,so that undulated noise is appeared on the background signal. However,the noise level is practically no problem in comparison with FIG. 4A.When σ=0.1 shown in FIG. 5B, the noise level is further reduced andexcellent contrast image can be obtained. According to the result, it isunderstood that the lower limit of σ is about 0.05. In order to securethe effect of the present invention, it is preferable to set the lowerlimit of σ to 0.1.

As shown above, although the accumulation effect on the backgroundsignal becomes higher as the value of σ becomes larger, the contrast ofthe image becomes lower as shown in FIGS. 5A through 5C, so that thevalue of σ cannot be made large imprudently. There must be an upperlimit. The upper limit of σ is considered below.

FIG. 6 is a graph showing contrast upon varying the value of σ where x=0as a solid line A. For the purpose of reference, there is another solidline B in FIG. 6 showing contrast where the same sample 5 is observed bythe bright field observation. The value of contrast in the bright fieldobservation becomes maximum 0.22 upon σ=0. Since higher contrast thanthe bright field observation is required, the value of σ showing thesame contrast value found from the graph is about 0.6, so that thisvalue becomes the upper limit of σ. In order to secure the effect of thepresent invention, it is preferable to set the upper limit of σ to 0.5.Accordingly, the contrast can be improved.

As a result, in a microscope apparatus according to the presentinvention, the following conditional expression (1) is preferablysatisfied:

0.05≦d1/(2×NA×f×m)≦0.6  (1)

where d1 denotes the aperture width of the slit aperture 3 a, NA denotesa numerical aperture of the objective lens 6, f denotes a focal lengthof the objective lens 6, m denotes the magnification from the rear focalplane of the objective lens 6 to the plane where the slit aperture 3 ais disposed in the illumination optical system 10.

In practical use, it is preferable that σ=0.4. However, it is notnecessary to stick on the condition upon emphasizing contrast or noisereduction effect, so that σ value may be selected within the scope ofconditional expression (1) in accordance with its use and purpose.

Second Embodiment

Then, a microscope apparatus according to a second embodiment of thepresent invention is explained with reference to FIGS. 7A, 7B, 7C, and8A through 8F. The microscope apparatus 100 according to the secondembodiment has the same configuration of the optical system of themicroscope apparatus 100 according to the first embodiment, and the onlydifference is that a portion of the π phase plate has a filter, whichcontrols transmittance, so that the explanation of the over allconfiguration is the same as that of the first embodiment. Accordingly,duplicated explanations are omitted.

FIGS. 7A, 7B and 7C are diagrams showing a π phase plate 17 with afilter having transmittance t disposed in a microscope apparatus 100according to a second embodiment, in which FIG. 7A shows construction ofthe π phase plate 17, FIG. 7B shows transmittance characteristics of theπ phase plate 17, and FIG. 7C shows phase characteristics of the π phaseplate 17.

In FIG. 7A, an outer circle 17 a of the π phase plate 17 denotes aneffective diameter of an objective pupil of the objective lens 6. Arectangular solid line in FIG. 7A shows a conjugate aperture of the slitaperture 13 a of the slit member 3, which is a projected image of theslit aperture 13 a on the plane of the π phase plate 17 and is denotedby the same symbol of 13 a. A filter 18 having transmittance t is formedto cover the slit aperture 18. Moreover, the filter 18 may be formed onthe π phase plate 17, or may be constructed independently and combinedintegrally with the π phase plate 17.

FIG. 7B shows transmittance distribution of the π phase plate 17 in theX axis direction corresponding to the objective pupil diameter 17 a, andtransmittance is decreased in a portion of the filter 18. FIG. 7C showsphase distribution of the π phase plate 17. The π phase plate 17 has aphase plate 17 e having a phase difference of −π/2 on the −X side withrespect to the Y axis, and a phase plate 17 f having a phase differenceof +π/2 on the X side with respect to the Y axis, and a case that thephase boundary 17 c, which is the boundary of the both plates, iscoincide with the Y axis is shown. In this case, the phase plate 17 ehas a phase difference of −π/2 and the phase plate 17 f has a phasedifference of +π/2, and the case the total phase difference is π isexplained. However, the present embodiment is not limited to theabove-described condition, and it is sufficient that light from thesample passing through the phase plate 17 f has a phase difference of π(180 degrees) with respect to light from the sample passing through thephase plate 17 e. In this manner, the π phase plate 17 is constructed.Similar to the first embodiment, the slit width d1 preferably satisfiesconditional expression (1).

Since transmittance t of the filter 18 covering the slit aperture 13 awith the slit width d1 is 50% or less, so-called direct light (0-orderdiffracted light) component is reduced as shown in FIG. 8A, so that thevisual field becomes dark in comparison with the first embodiment.However, the signal intensity becomes relatively strong with respect tothe background signal. As a result, contrast becomes higher than that ofthe first embodiment. In particular, contrast increase effect isconspicuous in a state where the shift amount in the X direction issmall (in the vicinity of x=0). Suitable transmittance may be chosen forthe filter 18 in accordance with its use and purpose.

FIGS. 8A through 8F are graphs showing imaging simulation results uponvarying the values of x and t where σ=0.1 in the π phase plate 17 shownin FIG. 7A. FIG. 8A is a case where t=10%, x=0, FIG. 8B is a case wheret=10%, X=0.4 mm shifted in the X direction. The simulation condition isthe same as the first embodiment. In comparison with FIG. 5, inparticular the case where x=0 (see FIGS. 5B and 8A), contrast increases.Imaging simulation results are respectively shown such that FIG. 8C isthe case where t=40%, x=0, FIG. 8D is a case where t=40%, x=0.4 mm, FIG.8E is a case where t=50%, x=0 mm, and FIG. 8F is a case where t=50%,x=0.4 mm. From these figures, it is understood that although lightintensity of the background increases and contrast relatively becomeslower as the transmittance t increases, even in the case where t=50%,contrast is sufficiently secured for practical use.

Accordingly, transmittance t is preferably satisfies the followingconditional expression (2):

0≦t≦50 (unit: %)  (2)

In the second embodiment, a case where transmittance t is constantwithin the aperture width d1 of the slit aperture 13 a is explained. Asa variation of the second embodiment, a case where transmittance t isminimum at x=0 and increases as X value is apart from x=0 along the Xaxis, and transmittance t is symmetrical with respect to the Y axis maybe applicable. For example, transmittance t may be proportional to xvalue, proportional to sin²(x), or incremental with respect to x value.

Third Embodiment

Then, a microscope apparatus according to a third embodiment of thepresent invention is explained. Since the third embodiment differsconstruction of a slit member and a phase plate thereof from that of thefirst embodiment, and the other constructions are the same as the firstembodiment, so that only the phase plate and the aperture member areexplained.

FIG. 9A is a diagram showing a π phase plate 27 disposed in a microscopeapparatus according to the third embodiment, showing construction andpositional relation of an annular aperture 23 a formed on an apertureportion 3. In other words, in the third embodiment, an aperture member 3with an annular aperture 23 a having a width of d2 is disposed at theposition where the slit member 3 is disposed in the FIG. 1, and a πphase plate 27 having a phase plate 27 e introducing phase difference of−π/2 with a disc shape, and, in the outer circle side thereof, a phaseplate 27 f introducing phase difference of +π/2 with a disc shape isdisposed in the vicinity of the rear focal point of the objective lens6. FIG. 9B shows transmittance characteristics of the π phase plate 27,and FIG. 9C shows phase characteristics of the π phase plate 27.Although a case that the phase plate 27 e has phase difference of −π/2,and the phase plate 27 f has phase difference of +π/2 is explained,since the phase difference of both phase plates is good enough to be π,the present invention is not limited to the above-describedconstruction.

In FIGS. 9A, 9B and 9C, the circular phase boundary portion 27 c, whichis a boundary between the phase plate 27 e and the phase plate 27 f, isdisposed to come to about the center of the annular aperture 23 a havingthe annular width d2 disposed the conjugate position of the π phaseplate 27 of the illumination optical system 10.

An image obtained by a microscope apparatus 100, in which the π phaseplate 27 and the annular aperture 23 a are disposed, is similar to theone obtained in the first embodiment at x=0. Accordingly, thecalculation result of the image is omitted. Although the image in thefirst embodiment has directional characteristics that the image hasresolution only in the direction of the aperture width d1 (in otherwords, in X direction), since the sample 5 is illuminated annularly bythe annular aperture 23 a in the third embodiment, the image of thethird embodiment does not have directional characteristics. Accordingly,the obtained two-dimensional image becomes a so-called edge-enhancedimage.

The aperture width d2 of the annular aperture 23 a and the aperturewidth d1 of the slit aperture 3 a of the first embodiment satisfy thefollowing relation:

d2=d1/2.

As a result, the third embodiment preferably satisfies the followingconditional expression (3) corresponding to conditional expression (1)of the first embodiment:

0.025≦d2/(2×NA×f×m)≦0.3  (3).

Conditional expression (3) defines the same meaning of the firstembodiment, so that duplicated explanations are omitted. In order tosecure the effect of the present invention, it is preferable to set thelower limit of conditional expression (3) to 0.05. In order to securethe effect of the present invention, it is preferable to set the upperlimit of conditional expression (3) to 0.25. In order to further securethe effect of the present invention, it is most preferable to set theupper limit of conditional expression (3) to 0.20.

FIGS. 10A, 10B and 10C shows a variation of the third embodiment. FIG.10A shows a π phase plate 37. FIG. 10B shows transmittancecharacteristics of the π phase plate 37. FIG. 10C shows phasecharacteristics.

In FIGS. 10A, 10B and 10C, a filter 38 having transmittance t is formedsuch that the π phase plate 37 has lower transmittance t at the annularaperture 23 a having the annular width d2 than other portions. Thefilter 38 having transmittance t may be formed on the π phase plate 37,or the filter 38 may be constructed independently and combinedintegrally with the π phase plate 37.

Moreover, transmittance t preferably satisfies the following conditionalexpression (2) as same as the first embodiment:

0≦t≦50 (unit: %)  (2).

In this manner, with disposing filter 38 having transmittance t on theannular aperture 23 a with the annular width d2, since the backgroundlight of the obtained sample image becomes dark and the signal lightbecomes relatively strong such as the second embodiment, the contrastbecomes higher in comparison with the case that the area of thetransmittance t is not exist such as FIG. 9.

Transmittance t of the filter portion 38 may be constant over theannular width d2 as shown above, or may be such that transmittance tbecomes minimum at the boundary 37 c of the phase difference and becomesgradually higher symmetrically with the boundary 37c. In this case,variation in transmittance t may be proportional to |r−rc| where rcdenotes the radius of the boundary area 37, or proportional tosin2(|r−rc|), or becomes gradually higher in accordance with |r−rc|.

Moreover, the phase plate 27, 37 are constructed movable in X, Y and Zdirections, and the effect thereof is the same as the first embodiment.

Fourth Embodiment

Then, a microscope apparatus according to a fourth embodiment of thepresent invention is explained. FIG. 11 is a schematic diagram showingthe microscope apparatus according to the fourth embodiment of thepresent invention. The point where the fourth embodiment differs fromthe first embodiment through the third embodiment, is that the aperturemember and the π phase plate are one each in the first through thirdembodiments, however, in the fourth embodiment, there are a plurality ofapertures in the aperture member, and each aperture is movable to theoptical axis of the illumination optical system, and there is a phaseplate holder having a plurality of π phase plates corresponding torespective apertures movable to the optical axis of the imaging opticalsystem. The same construction as the first embodiment is attached thesame symbol as the first embodiment, and duplicated explanations areomitted.

In FIG. 11, a microscope apparatus 200 according to the fourthembodiment has a slider-type aperture member 53 disposed in theillumination optical system 10 on the conjugate position of the rearfocal plane of the objective lens 6, and a slider-type phase plateholder 57 disposed in the vicinity of the rear focal plane of theobjective lens 6 in the imaging optical system 30. The slider-type phaseplate holder 57 may be disposed in the vicinity of the conjugate planeof the rear focal plane of the objective lens 6 in the imaging opticalsystem 30. The slider-type aperture member 53 may be disposed in thevicinity of the conjugate plane of the front focal plane of thecondenser lens 4 in the illumination optical system 10.

In the slider-type aperture member 53, slit apertures 3 a, 13 a, and anannular aperture 23 a are disposed substantially on the same plane andmovable to the optical axis of the illumination optical system 10 asshown in FIG. 12B. The aperture 3 a is the same one explained in thefirst embodiment, the aperture 13 a is in the second embodiment, and theaperture 23 a is in the third embodiment, so that explanations regardingconstruction are omitted.

In the slider-type phase plate holder 57, the π phase plate 7 used inaccordance with the slit aperture 3 a, the π phase plate 17 with afilter 18 used in accordance with the slit aperture 13 a, and the πphase plate 27 used in accordance with the annular aperture 23 a aredisposed substantially on the same plane interchangeable with respect tothe optical axis as shown in FIG. 12A. The π phase plate 7 is thesimilar one explained in the first embodiment, the π phase plate 17 isthe similar one explained in the second embodiment, and the π phaseplate 27 is the similar one explained in the third embodiment, so thatduplicated explanations are omitted. Moreover, respective π phase plates7, 17 and 27 are held by a holding member 57 a, which is equipped with afine adjuster 58 for finely adjusting the holding member 57 a in the Xdirection and a fine adjuster 59 for finely adjusting the holding member57 a in the Y direction in order to finely move the holding member 57 ain XY directions perpendicular to the optical axis. Furthermore, theslider-type phase plate holder 57 is constructed movable even in Zdirection for corresponding to variation in the rear focal plane uponchanging the objective lens 6.

In the fourth embodiment, since the slider-type aperture member 53 andthe slider-type phase plate holder 57 are constructed as describedabove, the optimum observation method corresponding to respectivesamples can be selected by changing combination of the aperture member 3a, 13 a, or 23 a with the π phase plate 7, 17 or 27. The holding member57 a of the phase plate holder 57 is movable in XY directions to adjustcontrast of the image. In the fourth embodiment, although theslider-type aperture member 53 and the slider-type phase plate holder 57are explained for changing, the other methods for changing such as aturret type for changing by rotation can be applied.

Moreover, when the aperture member is a slit aperture 3 a or 13 a, theslit width d1 satisfies conditional expression (1), and when theaperture member is the annular aperture 23 a, the aperture width d2satisfies conditional expression (3), and transmittance t of the filter18 satisfies conditional expression (2).

FIG. 13 and FIGS. 14A, 14B and 14C are diagrams showing a variation ofthe fourth embodiment of the present invention. FIG. 13 shows amicroscope apparatus according to the variation. FIG. 14A shows a phaseplate, FIG. 14B shows a filter, and FIG. 14C shows a state combined bothof them. In the variation, the π phase plate 17 and the filter 18 forvarying transmittance t are independently movable to the optical axis.The method for changing the aperture 3 a, 13 a and 23 a is the same asdescribed above.

In FIGS. 13, 14A, 14 b and 14C, the slider-type filter member 19 havinga filter 18 with transmittance t and the slider-type π phase plate 20having the π phase plate 17 are removably disposed in the optical axisof the imaging optical system 30. The slider-type filter member 19 andthe slider-type π phase plate 20 are removably disposed in the vicinityof the rear focal plane of the objective lens 6. The slider-type filtermember 19 and the slider-type π phase plate 20 have the sameconstruction as the second embodiment, so that the duplicatedexplanations of the operation and the effect are omitted.

In the present variation, the case that the slit aperture 13 a isinserted in the optical axis of the illumination optical system 10, andthe slider-type π phase plate 20 is inserted in the optical axis of theimaging optical system 30 is explained. When only the slider-type πphase plate 20 is inserted in the optical path, the microscope apparatus100 according to the first embodiment is constructed, and, in addition,when the slider-type filter member 19 is further inserted in the opticalaxis of the imaging optical system 30, the π phase plate becomes the onehaving characteristics of the slider-type π phase plate 20 together withcharacteristics of the slider-type filter member 19, so that themicroscope apparatus 100 according to the second embodiment can berealized. With providing phase plates having slightly differenttransmittance distribution or slightly different phase distribution, andby changing these phase plates, it becomes possible to realize variousobservation conditions.

Furthermore, when the slider-type filter member 19 and the slider-type πphase plate 20 are replaced by the slider-type filter member and theslider-type π phase plate each corresponding to the aperture shapedisposed on the slider-type aperture 53, the similar effect asabove-described each embodiment can be obtained.

Moreover, when the aperture member is a slit aperture 3 a or 13 a, theslit width d1 satisfies conditional expression (1), and when theaperture member is the annular aperture 23 a, the aperture width d2satisfies conditional expression (3), and transmittance t satisfiesconditional expression (2).

In the above-described each embodiment, although a transmission typemicroscope is explained, the same effect can be obtained by a reflectiontype microscope. In a reflection type microscope, when asemi-transparent mirror is used together with the illumination opticalsystem and the imaging optical system, an aperture member having anaperture or a slider-type aperture member is necessary to be disposedsubstantially conjugate position of the rear focal plane of theobjective lens between a light source of the illumination optical systemand the semi-transparent mirror, and a π phase plate or a slider-typephase plate holder or a filter member is necessary to be disposedsubstantially conjugate position of the rear focal plane of theobjective lens between the semi-transparent mirror and the image plane.

The present embodiment only shows a specific example for the purpose ofbetter understanding of the present invention. Accordingly, it isneedless to say that the invention in its broader aspect is not limitedto the specific details and representative devices shown and describedherein, and various modifications may be made without departing from thespirit or scope of the general inventive concept as defined by theappended claims and their equivalents.

1. A microscope apparatus comprising: an illumination optical systemthat illuminates a sample with illumination light emitted from a lightsource; an imaging optical system that converges light emitted from thesample to form a sample image by an objective lens; an aperture memberthat is disposed in the illumination optical system in the vicinity of aconjugate plane of a rear focal plane of the objective lens and has anaperture for limiting the illumination light; and a filter member thatincludes a phase plate that is disposed in the imaging optical system inthe vicinity of the rear focal plane of the objective lens or in thevicinity of the conjugate plane of the rear focal plane of the objectivelens and has a first phase area introducing phase difference of 180degrees into the light from the sample and a second phase area; a phaseboundary portion between the first phase area and the second phase areabeing disposed in a conjugate aperture of the aperture.
 2. Themicroscope apparatus according to claim 1, wherein the aperture is aslit aperture, and the phase boundary portion is substantially parallelto a long sides direction of the slit aperture.
 3. The microscopeapparatus according to claim 2, wherein the following conditionalexpression is satisfied:0.05≦d1/(2×NA×f×m)≦0.6 where d1 denotes a short side width of the slitaperture, NA denotes a numerical aperture of the objective lens, fdenotes a focal length of the objective lens, and m denotesmagnification from the rear focal plane of the objective lens to theplane in the illumination optical system where the slit aperture isdisposed.
 4. The microscope apparatus according to claim 3, wherein thefilter member further includes a transmittance controlling plate thatcontrols transmittance of the phase plate at the conjugate position ofthe slit aperture, and the transmittance controlling plate hassubstantially constant transmittance over the conjugate position of theslit aperture, and transmittance t satisfies the following conditionalexpression:0≦t≦50 (unit: %).
 5. The microscope apparatus according to claim 4,wherein the filter member includes a plurality of phase plates and aplurality of transmittance controlling plates, and the plurality ofphase plates and the plurality of transmittance controlling plates areindependently changeable with respect to the optical axis.
 6. Themicroscope apparatus according to claim 5, wherein the filter member ischangeable with respect to the optical axis.
 7. The microscope apparatusaccording to claim 3, wherein, in the phase plate, when the phaseboundary is assumed to be the Y axis, the axis perpendicular to the Yaxis and the optical axis is X axis, and the point of intersection ofthe X axis and the Y axis is to be an origin, the filter member furtherincludes a transmittance controlling plate that has transmittancedistribution such that transmittance of the filter member becomesminimum at the origin, and higher as being away from the origin, and thetransmittance distribution is symmetrical with respect to the Y axis. 8.The microscope apparatus according to claim 3, wherein, in the phaseplate, when the phase boundary is assumed to be the Y axis, the axisperpendicular to the Y axis and the optical axis is X axis, and thepoint of intersection of the X axis and the Y axis is to be an origin,the filter member further includes a transmittance controlling platethat has transmittance distribution such that transmittance of thefilter member becomes higher step-by-step as being away from the origin,and the transmittance distribution is symmetrical with respect to the Yaxis.
 9. The microscope apparatus according to claim 3, wherein thefilter member is changeable with respect to the optical axis.
 10. Themicroscope apparatus according to claim 2, wherein the filter memberfurther includes a transmittance controlling plate that controlstransmittance of the phase plate at the conjugate position of the slitaperture, and the transmittance controlling plate has substantiallyconstant transmittance over the conjugate position of the slit aperture,and transmittance t satisfies the following conditional expression:0≦t≦50 (unit: %)
 11. The microscope apparatus according to claim 2,wherein, in the phase plate, when the phase boundary is assumed to bethe Y axis, the axis perpendicular to the Y axis and the optical axis isX axis, and the point of intersection of the X axis and the Y axis is tobe an origin, the filter member further includes a transmittancecontrolling plate that has transmittance distribution such thattransmittance of the filter member becomes minimum at the origin, andhigher as being away from the origin, and the transmittance distributionis symmetrical with respect to the Y axis.
 12. The microscope apparatusaccording to claim 2, wherein, in the phase plate, when the phaseboundary is assumed to be the Y axis, the axis perpendicular to the Yaxis and the optical axis is X axis, and the point of intersection ofthe X axis and the Y axis is to be an origin, the filter member furtherincludes a transmittance controlling plate that has transmittancedistribution such that transmittance of the filter member becomes higherstep-by-step as being away from the origin, and the transmittancedistribution is symmetrical with respect to the Y axis.
 13. Themicroscope apparatus according to claim 1, wherein, in the phase plate,when the phase boundary is assumed to be the Y axis, the axisperpendicular to the Y axis and the optical axis is X axis, and thepoint of intersection of the X axis and the Y axis is to be an origin,the filter member further includes a transmittance controlling platethat has transmittance distribution such that transmittance of thefilter member becomes minimum at the origin, and higher as being awayfrom the origin, and the transmittance distribution is symmetrical withrespect to the Y axis.
 14. The microscope apparatus according to claim1, wherein, in the phase plate, when the phase boundary is assumed to bethe Y axis, the axis perpendicular to the Y axis and the optical axis isX axis, and the point of intersection of the X axis and the Y axis is tobe an origin, the filter member further includes a transmittancecontrolling plate that has transmittance distribution such thattransmittance of the filter member becomes higher step-by-step as beingaway from the origin, and the transmittance distribution is symmetricalwith respect to the Y axis.
 15. The microscope apparatus according toclaim 1, wherein the aperture of the aperture member is an annularaperture, the phase boundary portion of the phase plate is circular, andthe phase boundary portion is disposed substantially the center of anannular aperture conjugate with the annular aperture.
 16. The microscopeapparatus according to claim 15, wherein the following conditionalexpression is satisfied:0.025≦d2/(2×NA×f×m)≦0.6 where d2 denotes an aperture width of theannular aperture, NA denotes a numerical aperture of the objective lens,f denotes a focal length of the objective lens, and m denotesmagnification from the rear focal plane of the objective lens to theplane in the illumination optical system where the annular aperture isdisposed.
 17. The microscope apparatus according to claim 16, whereinthe filter member further includes a transmittance controlling platethat controls transmittance of the phase plate at the conjugate positionof the annular aperture, and the transmittance controlling plate hassubstantially constant transmittance over the conjugate position of theannular aperture, and transmittance t satisfies the followingconditional expression:0≦t≦50 (unit: %).
 18. The microscope apparatus according to claim 17,wherein the filter member includes a plurality of phase plates and aplurality of transmittance controlling plates, and the plurality ofphase plates and the plurality of transmittance controlling plates areindependently changeable with respect to the optical axis.
 19. Themicroscope apparatus according to claim 18, wherein the filter member ischangeable with respect to the optical axis.
 20. The microscopeapparatus according to claim 16, wherein the filter member furtherincludes a transmittance controlling plate that has transmittancedistribution concentric with respect to the optical axis, and theconcentric transmittance distribution is such that transmittance becomesminimum at an annular aperture position substantially conjugate with theannular aperture of the phase plate, becomes higher step-by-step asbeing away from the annular aperture position substantially conjugatewith the annular aperture of the phase plate, and is substantiallysymmetrical between a direction from an inner circumference of theaperture conjugate with the annular aperture to the center direction ofthe annular aperture and a direction from an outer circumference of theaperture conjugate with the annular aperture to the outer direction. 21.The microscope apparatus according to claim 16, wherein the filtermember is changeable with respect to the optical axis.
 22. Themicroscope apparatus according to claim 15, wherein the filter memberfurther includes a transmittance controlling plate that controlstransmittance of the phase plate at the conjugate position of theannular aperture, and the transmittance controlling plate hassubstantially constant transmittance over the conjugate position of theannular aperture, and transmittance t satisfies the followingconditional expression:0≦t≦50 (unit: %).
 23. The microscope apparatus according to claim 15,wherein the filter member further includes a transmittance controllingplate that has transmittance distribution concentric with respect to theoptical axis, and the concentric transmittance distribution is such thattransmittance becomes minimum at an annular aperture positionsubstantially conjugate with the annular aperture of the phase plate,becomes higher step-by-step as being away from the annular apertureposition substantially conjugate with the annular aperture of the phaseplate, and is substantially symmetrical between a direction from aninner circumference of the aperture conjugate with the annular apertureto the center direction of the annular aperture and a direction from anouter circumference of the aperture conjugate with the annular apertureto the outer direction.